Purification and characterization of anthranilate synthase component I (TrpE) from Mycobacterium tuberculosis H37Rv.
The emergence of multi-drug resistant (MDR) strains of Mycobacterium tuberculosis is the main reason why tuberculosis (TB) continues to be a major health problem worldwide. It is urgent to discover novel anti-mycobacterial agents based on new drug targets for the treatment of TB, especially MDR-TB. Tryptophan biosynthetic pathway, which is essential for the survival of M. tuberculosis and meanwhile absent in mammals, provides potential anti-TB drug targets. One of the promising drug targets in this pathway is anthranilate synthase component I (TrpE), whose role is to catalyze the conversion of chorismate to anthranilate using ammonia as amino source. In order to get a deep understanding of TrpE, a study on purification and characteristic identification of TrpE is required. In this work, the putative trpE gene of M. tuberculosis H37Rv was expressed as a fusion protein with a 6x His-tag on the N-terminal (His-TrpE) in Escherichia coli. The recombinant TrpE protein was successfully purified and then its enzymatic characteristics were analyzed. The native TrpE without His-tag was obtained by removal of the N-terminal fusion partner of His-TrpE using enterokinase. It was found that N-terminal fusion partner had little influence on TrpE catalytic activity. In addition, the key residues related to enzyme catalytic activity and that involved in l-tryptophan inhibition were predicted in the structure of M. tuberculosis H37Rv TrpE. These results would be beneficial to the designing of novel anti-TB drugs with high potency and selectivity.
Lin
Xiaohua
X
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, PR China.
Xu
Shengfeng
S
Yang
Yanping
Y
Wu
Junchen
J
Wang
Hongjun
H
Shen
Hongbo
H
Wang
Honghai
H
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000878:Anthranilate Synthase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000995:Antitubercular Agents / Q000494:pharmacology; D017124:Conserved Sequence; D015195:Drug Design; D024901:Drug Resistance, Multiple, Bacterial / Q000187:drug effects / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial / Q000187:drug effects; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014376:Tuberculosis / Q000235:genetics / Q000628:therapy; D018088:Tuberculosis, Multidrug-Resistant / Q000235:genetics
D000900:Anti-Bacterial Agents; D000995:Antitubercular Agents; D011994:Recombinant Proteins; D000878:Anthranilate Synthase